Application Note LCMS-108 Quantitation of benzodiazepines and Z-drugs in serum with the EVOQ TM LC triple quadrupole mass spectrometer

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1 Application Note LCMS-108 Quantitation of benzodiazepines and Z-drugs in serum with the EVOQ TM LC triple quadrupole mass spectrometer Abstract This study demonstrates a sensitive, rapid and reliable research method for the simultaneous quantitation of 18 benzodiazepines, metabolites and z-drugs in serum using the Bruker Advance TM UHPLC coupled to the EVOQ Elite TM triple quadrupole MS. Sample preparation was performed with liquid-liquid extraction. The method showed good precision and accuracy and a round robin test demonstrated excellent z-scores. Introduction Benzodiazepines (Figure 1) and the so called z-drugs are among the most subscribed drugs as they have sedative, hypnotic, anxiolytic, anticonvulsant and muscle relaxant properties but they also bear the risk of dependence. Traditionally they have been analyzed with gas chromatography or liquid chromatography (LC) with UV detector. LC coupled to a triple quadrupole mass spectrometer is now more commonly used as this technique provides increased selectivity and sensitivity and no derivatization is required. Authors Rafaela Martin, Karin Wendt Bruker Daltonik GmbH, Bremen, Germany Keywords Forensic Toxicology Benzodiazepines Z-drugs Quantitation Instrumentation and Software EVOQ LC-TQ Advance UHPLC

2 Benzodiazepines Figure 1: Basic structure of benzodiazepines Experimental Samples Calibrators were prepared by spiking blank serum with stock solutions of the analytes in the concentrations ranges indicated in Table 2. Furthermore, three commercial quality control samples containing different analytes in varying concentrations and two samples of a round robin test organized by the Society of Toxicological and Forensic Chemistry (GTFCh) with 13 of the analytes were analyzed. Sample Preparation 500 µl serum samples were incubated for 15 min at room temperature with 1.5 ml chloroform/isopropanol (95+5 v/v) and isotopic labelled internal standards (see Table 2) using a rotary mixer. After centrifugation the lower phase was transferred into an HPLC vial containing 50 µl 0.2 M hydrochloric acid in methanol. The organic solvent was evaporated and the residue reconstituted in 500 µl methanol/water (1+9 v/v). Instrumentation Instrumentation Liquid chromatography Instrument: Bruker Advance TM UHPLC Column: Acclaim RSLC 120, C18, 2.2 µm, 2.1 x 100 mm Mobile phase A: 1 % acetonitrile, 0.1 % formic acid with 2 mm ammonium formate Mobile phase B: 99 % acetonitrile, 0.1 % formic acid with 2 mm ammonium formate Gradient: min 1 % B min to 99 % B min 99 % B min to 1 % B min 1 % B Flow rate: 500 µl/min Injection volume: 1 µl Column oven: 40 C Mass Spectrometry Instrument: EVOQ Elite TM triple quadrupole mass spectrometer Ion source: VIP H-ESI positive, 4500 V Probe gas: 50 units at 400 C Cone gas: 25 units at 350 C Nebulizing gas: 50 units Active exhaust: on Collision gas: Argon, 1.5 mtorr MRM transitions: see Table 1 Results and Discussion Calibration curves showed good linearity (r² 0.994, Table 2). Replicate injections (n = 4) of all calibrators demonstrated excellent precision with RSD % for all analytes and accuracy was good with bias < ± 13% (20 % for the 100 ng/ml calibrator of midazolam). Detailed results for the three quality control samples are shown in Tables 3 5. The LODs (S/N 3) of ng/ml (Table 2) illustrate the sensitivity of the instrument. As the injection volume is only 1 µl and there is no preconcentration during sample preparation, the analysis could also be performed on limited sample volumes. The suitability of the method was demonstrated by passing a round robin test for all 13 analytes included in the study. The z-scores ((actual value target value)/standard deviation) were well below 1 (except for clonazepam 1.3) with acceptance criteria for passing the test of a z-score 2 (Table 6). Figure 2: EVOQ TM triple quadrupole MS with Advance TM UHPLC

3 Table 1: MRM transitions Rt (min) Precursor Ion Product Ion 1 CID 1 (V) Product Ion 2 CID 2 (V) 7-Aminoclonazepam Aminoflunitrazepam alpha-hydroxyalprazolam alpha-hydroxytriazolam Alprazolam Bromazepam Clonazepam Diazepam Flunitrazepam Flurazepam Lorazepam Midazolam Nordazepam Oxazepam Temazepam Zaleplon Zolpidem Zopiclone Chromatographic separation Figure 3: MRM traces for all analytes in calibrator 5/50 ng/ml

4 Table 2: Concentrations ranges and r² of calibrations; LODs Internal Standard Calibration Range r² LOD 7-Aminoclonazepam 7-Aminoclonazepam D Aminoflunitrazepam 7-Aminoflunitrazepam D alpha-hydroxyalprazolam alpha-hydroxyalprazolam D alpha-hydroxytriazolam alpha-hydroxyalprazolam D Alprazolam Alprazolam D Bromazepam alpha-hydroxyalprazolam D Clonazepam Nordazepam D Diazepam Diazepam D Flunitrazepam Flunitrazepam D Flurazepam Zaleplon D Lorazepam Oxazepam D Midazolam Zolpidem D Nordazepam Nordazepam D Oxazepam Oxazepam D Temazepam Nordazepam D Zaleplon Zaleplon D Zolpidem Zolpidem D Zopiclone Zopiclone D Table 3: Results for Quality control 1 Table 4: Results for Quality control 2 Bias [%] RSD [%] (n=4) Bias [%] RSD [%] (n=4) Diazepam Midazolam Nordazepam Midazolam Nordazepam Oxazepam Oxazepam

5 Table 5: Results for Quality control 3 Bias [%] RSD [%] (n=4) Midazolam Nordazepam Oxazepam Temazepam Table 6: Results of the round robin test Target value z-score 7-Aminoflunitrazepam Alprazolam Bromazepam Clonazepam Diazepam Flunitrazepam Lorazepam Midazolam Nordazepam Oxazepam Temazepam Zolpidem Zopiclone Conclusion The Bruker Advance TM UHPLC coupled to the EVOQ Elite TM triple quadrupole MS provides a sensitive solution for the rapid quantitation of benzodiazepines and z-drugs in serum. Linearity of calibration, precision and accuracy were excellent, with all analytes passing the round robin test, further demonstrating the general suitability of this quantitative workflow.

6 Bruker Daltonics is continually improving its products and reserves the right to change specifications without notice. Bruker Daltonics , LCMS-108, For research use only. Not for use in diagnostic procedures. Bruker Daltonik GmbH Bruker Daltonics Inc. Bremen Germany Phone +49 (0) Fax +49 (0) Billerica, MA USA Phone +1 (978) Fax +1 (978)